Duchenne Muscular Dystrophy (DMD) in both humans and dogs is a fatal, X-linked, recessive muscle disease caused by lack of dystrophin due to deletions or mutations in the dystrophin gene. Adeno- associated virus (AAV)-mediated delivery of a shortened (micro) dystrophin gene to skeletal muscle has been successful in mice, however, recent studies indicate that the efficacy of AAV-mediated therapies might be limited by an immune response to viral capsid proteins in humans. Similarly, we have demonstrated robust cellular immune responses to AAV capsid proteins following direct intramuscular injection of AAV vectors in wild type and cxmd (canine X-linked muscular dystrophy) dogs. Fortunately, we have demonstrated that this immune response can be averted by a brief course of immunosuppression. Furthermore, we have shown that previously used AAV vectors can transfer and express the AAV cap gene at low levels, and have found a method to prevent such transfer. Our results to date indicate that this and other vector modifications can reduce AAV vector immunogenicity in dogs. The broad, long-term objective of this project is to develop AAV-mediated gene therapy strategies in cxmd dogs that can be applied to human patients with DMD. We are now ready to test an optimized AAV vector that expresses micro- dystrophin in humans. In this revision application, we are requesting funding for production of the clinical-grade AAV vector and for costs associated with regulatory approval required for clinical studies. Drs. Miller and Halbert at the FHCRC, who have participated in previous clinical trials of gene therapy and have the required vector production and regulatory approval experience, will spearhead these efforts. We plan to carry out initial clinical trials in parallel with additional studies in dogs, conducted as part of the parent application, to refine and improve techniques for effective treatment of DMD in humans. PUBLIC HEALTH RELEVANCE: The importance of this revision application is that it will provide funding to make clinical-grade AAV vectors designed to treat Duchenne muscular dystrophy (DMD), and to obtain regulatory approval for testing of these vectors in humans. Our current studies are at the point where we are ready to test an optimized AAV vector in humans, and funding for clinical vector production and for regulatory approval is critically required to pursue our goal of effective gene therapy for human DMD.